Inotodiol prevents age-related muscle wasting by restoring mitochondrial function through liver X receptor beta signaling

Abstract

Sarcopenia is characterized by a progressive decline in muscle mass and strength and represents a major contributor to increased mortality in the elderly population. Mitochondrial dysfunction, which leads to impaired energy metabolism and elevated oxidative stress, is a key driver of muscle wasting and associated metabolic disorders. Thus, mitochondrial targeting is a promising strategy for combating sarcopenia. In this study, we investigated the therapeutic potential of the mycosterol inotodiol (Ino) in mitigating age-related muscle wasting and metabolic dysfunction. Ino treatment significantly improved muscle mass and function in aged mice and prevented dexamethasone (DEX)-induced muscle atrophy. Ino enhanced mitochondrial function and restored muscle metabolism, as evidenced by increased mitochondrial content, elevated oxidative capacity, reduced lipid accumulation, and decreased oxidative stress. In addition, Ino attenuated palmitic acid (PA)-induced lipotoxicity in muscle cells by restoring lipid metabolism. Further investigation revealed that Ino activates liver X receptor β (LXRβ) and promotes its interaction with peroxisome proliferator-activated receptor δ (PPARδ), thereby increasing sirtuin 3 (Sirt3) transcription. Consequently, Ino activates the LXRβ/SIRT3/peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) signaling pathway, a central regulator of mitochondrial function and metabolic homeostasis. Collectively, these findings identify Ino as a promising therapeutic candidate for preserving mitochondrial function and alleviating age-associated muscle dysfunction.

Introduction

Sarcopenia is a debilitating, age-associated condition characterized by the progressive loss of skeletal muscle mass, strength, and functional capacity. It is a major contributor to frailty, loss of independence, metabolic disorders, and increased mortality in the elderly population.^1–3 With the rapid expansion of aging societies worldwide, sarcopenia has emerged as an important public health concern with substantial clinical and socioeconomic consequences, imposing a growing burden on healthcare systems and significantly reducing the quality of life of affected individuals. The prevalence of sarcopenia continues to increase with advancing age, contributing to compromised mobility, falls, fractures, disability, and prolonged hospitalization, thereby imposing a substantial burden on healthcare system. Consequently, preserving skeletal muscle mass and metabolic function has become a major priority for promoting healthy aging and extending health span. However, despite extensive research efforts, the molecular mechanisms responsible for age-related muscle deterioration remain incompletely understood, and effective pharmacological therapies for sarcopenia are still lacking,^4,5 highlighting the urgent need to identify novel therapeutic targets and effective intervention strategies for preventing disease progression.

Mitochondrial dysfunction and disrupted lipid metabolism are increasingly recognized as central drivers of sarcopenia.^6–10 Excessive intramyocellular lipid accumulation, reduced oxidative capacity, impaired insulin sensitivity, and decreased mitochondrial content and function in aging skeletal muscle are consistently observed and are strongly associated with progressive muscle degeneration during aging.^11–14 These metabolic abnormalities contribute to energy deficiency, excessive reactive oxygen species production, and the activation of catabolic pathways, collectively promoting muscle weakness and functional decline.^15–18 Accumulating evidence further indicates that impaired mitochondrial quality control, including defects in mitochondrial biogenesis, dynamics, and mitophagy, accelerates the deterioration of muscle metabolic capacity during aging. Failure to maintain mitochondrial integrity disrupts ATP production and increases oxidative damage, creating a vicious cycle that exacerbates cellular dysfunction and muscle atrophy. Importantly, lipid metabolic disturbances and mitochondrial dysfunction are closely interconnected, as impaired fatty acid utilization can exacerbate oxidative stress and further compromise mitochondrial homeostasis. Conversely, defective mitochondrial oxidative phosphorylation limits lipid oxidation, leading to the accumulation of toxic lipid intermediates that further impair insulin signaling and metabolic flexibility in skeletal muscle. Several molecular regulators play pivotal roles in maintaining these processes in skeletal muscle. Among them, the mitochondrial deacetylase SIRT3 and the transcriptional coactivator PGC-1α are critical regulators of mitochondrial biogenesis, oxidative metabolism, and cellular stress resistance.^19–23 Together these regulators coordinate mitochondrial adaptation to energetic stress and are essential for preserving metabolic homeostasis and muscle function during the aging process.

In aging muscle, reduced expression of SIRT3 and PGC-1α has been associated with defective mitochondrial regulation, impaired oxidative stress responses, and abnormal lipid metabolism, ultimately contributing to lipid accumulation and metabolic dysfunction.^23–25 Consistent with these observations, studies in sarcopenic muscle have shown that mitochondrial dysfunction is accompanied by the suppression of anabolic signaling pathways and the development of anabolic resistance.^15,26,27 In addition to mitochondrial regulators, liver X receptors (LXRs), members of the nuclear receptor family, are key regulators of lipid and cholesterol metabolism.²⁸ While the liver X receptor α (LXRα) predominantly regulates hepatic lipid homeostasis, LXRβ seems to play a dominant role in skeletal muscle.^29,30 LXRs form heterodimers with other nuclear receptors, such as Retinoic X Receptors (RXRs) or Peroxisome Proliferator-Activated Receptors (PPARs), to modulate genes involved in lipid metabolism, glucose utilization, and energy homeostasis.^31–33 Beyond their metabolic functions, LXR agonists have demonstrated anti-inflammatory and cytoprotective effects in several disease models, including atherosclerosis,^33–35 suggesting broader roles in maintaining cellular homeostasis and protecting tissues from metabolic stress under pathological and age-related conditions. Emerging evidence further suggests that LXR signaling may influence mitochondrial quality control and oxidative metabolism through functional interactions with transcriptional regulators involved in energy homeostasis. However, the mechanisms linking lipid sensing to mitochondrial regulation in aging skeletal muscle remain poorly understood, and the potential contribution of LXRβ to this process has not been established. This knowledge gap has limited the development of mechanism-based therapeutic strategies aimed at restoring metabolic homeostasis and mitochondrial function in aging skeletal muscle. Therefore, identifying the molecular mechanisms by which LXRβ coordinates lipid metabolism and mitochondrial function may provide new opportunities for developing targeted therapies to combat sarcopenia and other age-related metabolic disorders.

Our previous study demonstrated that Inonotus obliquus (I. obliquus) extract improves muscle regeneration and attenuates DEX-induced muscle atrophy in association with the modulation of PGC-1α and mitochondrial-related pathways.³⁶ In the present study, we identified Ino, a major mycosterol component of I. obliquus, as a key bioactive compound that mediates these effects. We therefore investigated the effects of Ino on muscle metabolism, mitochondrial regulation, and muscle performance during aging. Our findings reveal that Ino alleviates muscle degeneration, enhances mitochondrial biogenesis and oxidative capacity, and mitigates lipid accumulation in aged skeletal muscles. Furthermore, we identify LXRβ as a previously unrecognized regulator of mitochondrial homeostasis and show that Ino exerts its protective effects through activation of the LXRβ/SIRT3/PGC-1α signaling pathway. Collectively, these results highlight Ino as a promising therapeutic candidate for combating age-related sarcopenia and other muscle wasting conditions.

Results

Muscle aging is associated with metabolic disturbances, mitochondrial dysfunction, and a decrease in the activity of the SIRT3/PGC-1α pathway

To gain insights into metabolic changes associated with muscle aging, young (Y) and aged (A) mouse muscles were subjected to metabolite profiling (Fig. 1a). Hierarchical clustering revealed aging-related alterations in diverse metabolites. Among the intramuscular metabolites, those related to fatty acid or amino acid metabolism were markedly altered, suggesting disturbed amino acid or fatty acid homeostasis.

Fig. 1.

Muscle aging is associated with metabolic disturbances, mitochondrial dysfunction, and a decrease in the activity of the SIRT3/PGC-1α pathway. a Untargeted metabolomic profiling of young and aged mouse skeletal muscle reveals age-associated shifts in metabolite abundance. The heatmap displays z score-scaled metabolite levels, and the side annotation indicates chemical classes. b Heatmap showing the GSEA-based metabolic pathways across the myonuclei. The color denotes the normalized enrichment score. c Heatmap showing the expression profiles of oxidative stress-related genes across myonuclei. Red arrows indicate the most prominent changes in expression patterns. d Quantitative analysis of myofiber cross-sectional area from H&E-stained sections in a human cohort derived from the GTEx database using a computational pipeline that measures fiber size in pixel units and converts values to micrometers using the recorded pixel size. Whole-slide images were acquired with recorded pixel sizes, and regions of interest were digitally magnified fourfold while the physical scale was preserved. Scale bar = 180 µm. e qRT‒PCR analysis of mRNAs related to mitochondrial metabolism in the gastrocnemius (GAS) muscles of 3-month-old (young) and 24-month-old (aged) male mice. Rn18s was used as an internal control. (n = 4 for young; n = 3 for aged). f Immunoblot analysis of PGC-1α and SIRT3 in total lysates or mitochondrial (Mito) fractions of GAS muscles from young and aged male mice. The quantification of the relative protein expression levels is shown on the right. Ponceau S or VDAC1 served as loading controls (n = 3 per group for total lysates; n = 4 for young mito; n = 3 for aged mito). g Relative ratio of mtDNA (Mt-co2) to nuclear DNA (nDNA, Gapdh) in GAS muscles from young and aged male mice (n = 4 per group). h Relative integrated density (IntDen) ratio of JC-1 aggregates (red) to monomers (green) in single myofibers isolated from the EDL muscles of young and aged male mice (n = 4 for young; n = 3 for aged; 3–6 myofibers analyzed per individual). Representative images are shown in Supplementary Fig. 1e. i Relative IntDen for mitoSOX fluorescence (red) in single myofibers isolated from the EDL muscles of young and aged male mice. Representative images are shown in Supplementary Fig. 1f (n = 4 for young; n = 3 for aged; 3–4 myofibers analyzed per individual). The data are expressed as the means ± SDs. Statistical significance was determined using an unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, and ***p < 0.001

Next, we analyzed publicly available single-nucleus RNA sequencing datasets of young and old muscle.³⁷ Young muscles exhibited a relatively even distribution across six distinct myonuclei clusters, whereas aged muscles lost this heterogeneity, indicating a marked shift toward cluster 1 (Supplementary Fig. 1a). Gene set enrichment analysis (GSEA)-based metabolic pathway analysis revealed that the oxidative phosphorylation scores of cluster 1 myonuclei markedly decreased, whereas the glycolysis-related scores remained relatively stable across all the clusters (Fig. 1b). Further reactome pathway analysis predicted alterations in creatine metabolism, phenylalanine metabolism, linoleic acid metabolism, lipid metabolism, and mitochondrial fatty acid metabolism in aged muscles compared with young muscles (Supplementary Fig. 1b). Consistent with these findings, biological process enrichment analysis of Cluster 1 myonuclei revealed low enrichment scores for gene sets related to mitochondrial membrane transport and ATP metabolic processes, suggesting a potential association with mitochondrial dysfunction (Supplementary Fig. 1c).

In the detailed analysis of oxidative stress-related genes, cluster 1 exhibited decreased Sirt3 expression, which was accompanied by increased expression of two muscle-specific E3 ligases, F-box protein 32 (Fbxo32) and tripartite motif containing 63 (Trim63), encoding Atrogin1 and Muscle Ring Finger Protein 1 (MuRF1), respectively (Fig. 1c). Analysis of genotype-tissue expression (GTEx) data from elderly human cohort samples revealed that compared with muscles with high SIRT3 expression, muscles with low SIRT3 expression displayed smaller myofiber cross-sectional areas (Fig. 1d). To assess whether SIRT3 expression is coordinated with the expression of other sirtuin family members, such as SIRT1, we analyzed SIRT1 expression in the same subjects. However, SIRT1 expression was not correlated with SIRT3 expression (Supplementary Fig. 1d). These findings suggest that reduced SIRT3 levels are associated with impaired muscle architecture, which is potentially linked to increased interstitial fibrosis.

To validate these findings, we conducted quantitative RT‒PCR (qRT‒PCR) on gastrocnemius (GAS) muscle samples from 4-month-old and 24-month-old mice. Aged muscle displayed significantly reduced expression of key mitochondrial regulators, including Sirt3, Ppargc1a, and peroxisome proliferator-activated receptor γ (Pparg), along with downregulation of genes involved in mitochondrial function, antioxidant defense, lipid metabolism, and glucose metabolism (Fig. 1e; Supplementary Fig. 1e). A consistent reduction in mitochondrial metabolism gene expression was observed when the expression was normalized to that of either 18S ribosomal RNA (Rn18s) or ribosomal protein L32 (Rpl32) (Fig. 1e; Supplementary Fig. 1f).

At the protein level, aged GAS muscles exhibited a marked reduction in PGC-1α and SIRT3 expression, which correlated with reduced mitochondrial content (Fig. 1f, g). Consistent with these findings, functional assays demonstrated significant mitochondrial dysfunction in aged extensor digitorum longus (EDL) muscles. JC-1 staining revealed a reduced mitochondrial membrane potential (Fig. 1h; Supplementary Fig. 1g), whereas MitoSOX staining indicated increased mitochondrial oxidative stress in aged myofibers compared with young myofibers (Fig. 1i; Supplementary Fig. 1h).

Collectively, these findings highlight the extensive metabolic dysregulation that accompanies muscle aging and underscore the critical role of mitochondrial homeostasis in preserving muscle health. Targeting mitochondrial function has emerged as a promising therapeutic strategy for combating sarcopenia, with the aim of enhancing mitochondrial biogenesis, oxidative capacity, and metabolic balance and ultimately mitigating age-related muscle degeneration.

Ino enhances myogenic differentiation and mitochondrial function in muscle cells

Considering the beneficial effects of I. obliquus extract on muscle mitochondrial function, we sought to identify the bioactive compounds responsible for inducing PGC-1α expression.³⁶ Among compounds derived from I. obliquus, we identified Ino as a potent activator of PGC-1α. C2C12 myoblasts transfected with a Ppargc1a-reporter construct were treated with increasing concentrations of Ino (0.1–10 μM) or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a positive control. Ino treatment induced Ppargc1a-reporter activity in a dose-dependent manner, peaking at 1 μM before decreasing at 10 μM (Fig. 2a). Notably, compared with AICAR at 0.5 mM, 1 μM Ino elicited stronger induction of Ppargc1a-reporter activity. Interestingly, PGC-1α protein levels increased progressively with Ino treatment, peaking at 10 μM (Fig. 2b). Importantly, none of the tested Ino concentrations affected cell viability (Fig. 2c; Supplementary Fig. 2a).

Fig. 2.

Ino elicits metabolic alterations characterized by enhanced mitochondrial activity in muscle cells. a Relative Ppargc1a promoter-driven luciferase activity in C2C12 cells treated with DMSO (-), Ino, or AICAR at the indicated concentrations for 24 h (n = 3 per group). b Immunoblot analysis of PGC-1α protein levels in C2C12 cells treated with the indicated concentrations of Ino for 24 h. The right graph shows the quantification of relative PGC-1α protein expression normalized to that of β-actin (n = 3 per group). c Relative MTT absorbances (570 nm) in C2C12 cells treated with the indicated concentrations of Ino for 24 h (n = 4 per group). d qRT‒PCR analysis of the mRNA expression of the indicated genes in C2C12 cells treated with Ino at the indicated concentrations for 24 h. Rn18s was used as an internal control (n = 3 per group). e Immunostaining for myosin heavy chain (MHC; red) in C2C12 cells treated with the indicated concentrations of Ino for 48 h. The nuclei were counterstained with DAPI (blue). f qRT‒PCR analysis of the mRNA expression of the indicated genes in C2C12 cells treated with Veh or 1 µM Ino for 24 h (n = 3 per group). g Immunoblot analysis of PGC-1α, SIRT3 and acetylated lysine levels in total lysates or mito fractions of C2C12 cells treated with Veh or 1 µM Ino for 24 h. The graphs on the right show the quantification of relative protein expression levels. β-Tubulin (total) or VDAC1 (Mito) was used as a loading control (n = 3 per group). h qRT‒PCR analysis of the mRNA expression of the indicated genes in C2C12 cells treated with Veh or 1 µM Ino for 24 h (n = 3 per group). i Immunoblot analysis of total OXPHOS complex proteins in C2C12 cells treated with Veh or 1 µM Ino for 24 h. The graph on the right shows the quantification of relative protein expression normalized to that of β-actin (n = 3 per group). j Relative ratio of mtDNA to nDNA in C2C12 cells treated with Veh or 1 µM Ino for 24 h (n = 3 per group). k Relative TMRM fluorescence intensity (Ex/Em = 548/573) in C2C12 cells treated with Veh or 1 µM Ino for 24 h (n = 4 per group). l Oxygen consumption rate (OCR) values in C2C12 cells treated with Veh or 1 µM Ino for 24 h were normalized to the mtDNA copy number (mtDNA/nDNA) and multiplied by 10³ for visualization (n = 3 per group). As shown in the graph, the indicated steps involved treatment with 1.5 µM oligomycin, 3 µM FCCP, and 0.5 µM rotenone (Rot)/antimycin A (AA). m Quantification of mitochondrial respiration parameters derived from the data shown in panel l (n = 3 per group). The data are presented as the mean ± SD. Statistical significance was determined using one-way ANOVA (panels a–d and k), unpaired two-tailed Student’s t test (panels f–j and m) or two-way ANOVA (panel l). *p < 0.05, **p < 0.01, and ***p < 0.001

In addition to PGC-1α induction, Ino promoted myogenic differentiation. The expression of key myogenic markers, myogenin (Myog) and troponin T1 (Tnnt1), progressively increased with Ino treatment, with the strongest induction observed at 1 μM (Fig. 2d). Myosin heavy chain (MyHC) immunostaining consistently revealed enhanced myotube formation, with the most pronounced effect observed at 1 μM Ino (Fig. 2e; Supplementary Fig. 2b, c). On the basis of these findings, 1 μM Ino was selected for subsequent experiments.

To further investigate the impact of Ino on metabolic and stress-related gene expression, C2C12 myoblasts were treated with Ino for two days in differentiation medium, followed by qRT‒PCR analysis. Ino treatment significantly upregulated PGC-1α and SIRT3 at the mRNA and protein levels (Fig. 2f, g). Consistently, the acetylation levels of mitochondrial proteins were reduced, which correlated with increased Sirt3 activity in the mitochondrial fraction of Ino-treated cells.

Furthermore, Ino treatment markedly upregulated the expression of genes involved in mitochondrial function, mitochondrial fatty acid metabolism, antioxidant defense, and glucose metabolism (Fig. 2h). Conversely, the expression of the muscle atrophy-associated genes myostatin (Mstn), Fbxo32 and Trim63 was significantly suppressed by Ino treatment (Supplementary Fig. 2d).

In agreement with the gene expression profiles, compared with vehicle-treated control myotubes, Ino-treated myotubes exhibited increased oxidative phosphorylation (OXPHOS) protein levels and mitochondrial content (Fig. 2i, j). These findings are further corroborated by the results showing elevated mitochondrial membrane potential in Ino-treated cells, as assessed by tetramethylrhodamine (TMRM) (Fig. 2k). Consistent with these observations, mitochondrial respiration was assessed using a Seahorse XF Analyzer following Ino treatment, and ATP-linked respiration, along with a trend toward increased carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)-stimulated respiration, was observed in Ino-treated cells, suggesting enhanced mitochondrial ATP production capacity and metabolic flexibility (Fig. 2l, m). Collectively, these findings indicate that Ino is associated with enhanced mitochondrial activity and differentiation-related features in muscle cells.

Ino treatment mitigates DEX-induced muscle wasting in young mice

DEX, a synthetic glucocorticoid, induces muscle atrophy by activating catabolic pathways and disrupting mitochondrial function.^38,39 To assess whether Ino could counteract these effects, C2C12 myotubes were differentiated for three days and treated for 24 h with DMSO (vehicle; Veh), Ino, and/or DEX (Supplementary Fig. 3a). Vehicular-treated myotubes displayed marked atrophy following DEX exposure, whereas Ino cotreatment effectively prevented myotube atrophy.

At the molecular level, DEX treatment significantly upregulated the expression of the muscle-specific E3 ligases Fbxo32 and Trim63. In contrast, Ino cotreatment markedly attenuated this induction (Supplementary Fig. 3b), as confirmed by protein analysis, which revealed reduced Atrogin-1 levels and restored MyHC protein levels (Supplementary Fig. 3c, d). Furthermore, Ino cotreatment modestly but significantly increased the phosphorylation of AKT serine/threonine kinase 1 (AKT1) and mechanistic target of rapamycin (MTOR), both of which were suppressed by DEX. In addition, Ino partially restored AMP-activated protein kinase α (AMPKα) phosphorylation, which was suppressed by DEX), and upregulated SIRT3 and PGC-1α protein levels, suggesting that Ino plays a role in maintaining mitochondrial function.

To validate these in vitro findings, we conducted an in vivo study using 9-week-old mice treated with DEX (20 mg/kg) and either vehicle or Ino (0.35 mg/kg) for 10 weeks. Ino cotreatment slightly attenuated DEX-induced body weight loss (Supplementary Fig. 4a), whereas food intake did not differ between the DEX and DEX + Ino groups (Supplementary Fig. 4b). Moreover, Ino attenuated DEX-induced reductions in grip strength and partially mitigated the decline in endurance exercise capacity (Supplementary Fig. 4c, d). Consistent with these functional effects, Ino treatment resulted in a trend toward partially preserved muscle mass in the GAS, tibialis anterior (TA), and EDL, although these differences did not reach statistical significance (Supplementary Fig. 4e, f). Epididymal white adipose tissue (eWAT) mass was not affected by Ino treatment (Supplementary Fig. 4g). Consistent with the effects on muscle weight, Ino treatment preserved the myofiber cross-sectional area (CSA) of TA muscle sections, which was reduced by DEX (Supplementary Fig. 4h).

Mechanistically, protein analysis of GAS muscles revealed that Ino partially reversed the DEX-induced suppression of AKT‒mTOR signaling and attenuated the upregulation of Atrogin-1 expression (Supplementary Fig. 4i, j). In line with our in vitro findings, compared with Veh, Ino increased SIRT3 and PGC-1α protein levels, supporting its role in enhancing mitochondrial function and muscle metabolic homeostasis. At the mRNA level, DEX-induced upregulation of Fbxo32 and Trim63 tended to decrease following Ino treatment, although the changes did not reach statistical significance (Supplementary Fig. 4k). Moreover, Ino preserved the mitochondrial content in DEX-treated GAS muscle (Supplementary Fig. 4l) and alleviated the DEX-induced decrease in the mitochondrial protein synthesis rate in quadriceps (QUA) muscles, further supporting its role in sustaining mitochondrial function (Supplementary Fig. 4m).

Collectively, these findings demonstrate that Ino protects against DEX-induced muscle atrophy by inhibiting catabolic pathways and preventing mitochondrial dysfunction.

Ino treatment enhances muscle mass, strength, and mitochondrial function in sedentary young mice

Considering that Ino activates pathways that regulate muscle metabolism, we investigated its effects on muscle mass, function, and mitochondrial activity in young adult mice. Three-month-old mice were administered vehicle or Ino (0.35 mg/kg) daily for eight weeks. Body weights remained comparable between the groups (Fig. 3a); however, compared with control mice, Ino-treated mice exhibited significantly increased food intake (Fig. 3b). Ino supplementation enhanced grip strength (Fig. 3c) and endurance exercise capacity (Fig. 3d). Moreover, Ino-treated mice displayed a mild but significant reduction in eWAT mass, whereas brown adipose tissue (BAT) mass increased (Fig. 3e). Notably, these functional improvements were accompanied by a modest but significant increase in muscle weight in Ino-treated mice (Fig. 3f), suggesting that Ino increases muscle mass and strength while concurrently reducing fat accumulation in sedentary young mice.

Fig. 3.

Ino treatment enhances muscle mass, strength, and mitochondrial function in sedentary young mice. a, b Three-month-old male mice were orally administered vehicle (Veh) or Ino daily for 2 months. Body weight (n = 9 per group) (a) and average daily food intake (n = 9 per group) (b) were measured. c Maximal grip strength of mice treated with Veh or Ino normalized to individual body weight (n = 9 for Veh; n = 7 for Ino). d Total running distance (left) and running time (right) measured using an inclined treadmill exercise test (n = 9 per group). e Relative fat mass normalized to body weight (n = 10 for Veh; n = 9 for Ino). f Relative hindlimb muscle weights (TA, EDL, GAS, and soleus (Sol) muscles) (n = 5 per group). g H&E staining of TA muscles. The graph on the right shows the quantification of the average CSA (µm²) (n = 4 per group). h qRT‒PCR analysis of the relative mRNA expression levels of genes associated with myofiber type in TA muscles from Veh- or Ino-treated young mice (n = 4 per group). i SDH and NADH-TR staining of TA muscles. The graphs on the right show the percentage of the area positive for SDH or NADH-TR enzyme activity (n = 9 fields for Veh; n = 7 fields for Ino). j qRT‒PCR analysis of the mRNA expression levels of oxidative phosphorylation (OXPHOS)-related genes in TA muscles from Veh- or Ino-treated young mice (n = 4 per group). k Immunoblot analysis of total OXPHOS complex proteins and the myoglobin levels of GAS muscles from Veh- or Ino-treated young mice. GAPDH was used as a loading control. The graph on the right presents the relative protein expression levels (n = 4 per group). l JC-1 staining of live single myofibers isolated from EDL muscles. The graph on the right shows the ratio of the IntDen of JC-1 aggregates (red) to that of monomers (green) (n = 3 per group; 5–6 myofibers analyzed per individual). m The ratio of relative mtDNA to nDNA in QUA muscles from Veh- or Ino-treated young mice (n = 4 per group). n Fractional synthesis rate (FSR) of mitochondrial proteins measured in heavy water (²H₂O)-labeled mitochondrial lysates of GAS muscles from Veh- or Ino-treated young mice (n = 10 per group). o OCR was measured in mitochondria isolated from the GAS muscle of Veh- or Ino-treated young mice (n = 3 per group; 10 μg of mitochondria per well). The indicated steps involved treatment with 1 mM ADP, 1.5 µM oligomycin, 2 µM FCCP, and 0.5 µM Rot/AA. p Quantification of mitochondrial respiration parameters derived from the data shown in panel o (n = 3 per group). The data are presented as the mean ± SD. Statistical significance was determined by an unpaired two-tailed Student’s t test unless otherwise indicated (panel o, two-way ANOVA). *p < 0.05, **p < 0.01, and ***p < 0.001

Histological analysis of TA muscles by hematoxylin and eosin (H&E) staining revealed a significant increase in myofiber CSA in Ino-treated muscles compared with controls (Fig. 3g). Gene expression profiling revealed increased expression of myosin heavy chain genes, including fast-twitch isoforms (Myh1, Myh2, and Myh4) and low-abundance Myh7, which is consistent with the upregulation of muscle structural gene expression rather than a fiber-type switch (Fig. 3h). In addition, Ino treatment enhanced oxidative metabolism, as evidenced by increased numbers of myofibers with stronger succinate dehydrogenase (SDH) and NADH-tetrazolium reductase (NADH-TR) activities in TA muscles (Fig. 3i). Consistent with these findings, mitochondrial gene expression was significantly upregulated in Ino-treated GAS muscles (Fig. 3j). Compared with vehicle treatment, Ino treatment elevated total OXPHOS and myoglobin levels at the protein level in GAS muscles (Fig. 3k). Additionally, JC-1 staining revealed a significant increase in the aggregate ratio in Ino-treated myofibers from EDL muscles, indicating increased mitochondrial membrane potential and activity (Fig. 3l). Consistently, Ino treatment elevated both the mitochondrial DNA (mtDNA) content in QUA muscles and mitochondrial protein synthesis rates in GAS muscles (Fig. 3m, n). To further assess mitochondrial function, we analyzed mitochondrial respiration in mitochondria isolated from GAS muscles (Fig. 3o, p). Basal and ATP-linked respiration did not differ between the groups, but intriguingly, Ino treatment increased FCCP-stimulated maximal respiration and spare capacity. These results suggest that Ino maintains basal mitochondrial function while improving metabolic flexibility in young muscle.

Collectively, these findings indicate that long-term Ino treatment is associated with improved muscle mass, strength, and function in sedentary young mice and is accompanied by changes in the expression of mitochondrial and oxidative metabolism–related markers.

Ino treatment prevents age-related muscle loss and weakness

Aging is associated with mitochondrial dysfunction and metabolic disturbances that contribute to muscle wasting.⁴⁰ To investigate whether Ino could protect against age-related muscle decline, twenty-month-old mice were treated with Ino (0.35 mg/kg) or vehicle daily for ten weeks (Supplementary Fig. 5a). While body weights were not significantly altered by Ino treatment, Ino-treated mice consumed more food than control mice did (Fig. 4a, b). Blood glucose levels and serum free fatty acid (FFA) levels were markedly reduced in Ino-treated mice (Fig. 4c, d).

Fig. 4.

Ino treatment prevents age-related muscle loss and weakness. a Twenty-month-old (aged) male mice were orally administered Veh or Ino daily for 7 weeks. Body weight was measured before (0 week) and after (7 weeks) Veh or Ino treatment prior to sacrifice (n = 10 for Veh; n = 9 for Ino). b Average daily food intake (n = 8 per group). c Blood glucose levels measured before (0 week) and after (7 weeks) Veh or Ino treatment (n = 10 for Veh; n = 9 for Ino). d FFA (mmol/L) levels in serum from Veh- or Ino-treated mice (n = 10 for Veh; n = 8 for Ino per group). e Treadmill running time (left) and grip strength (right). Treadmill running tests were performed without incline (n = 9 per group). Grip strength represents the mean of four repeated measurements per individual (n = 7 for Veh; n = 8 for Ino). f Representative electromyography (EMG) trace graph of the FDB muscles of Veh- or Ino-treated mice. g Quantification of the EMG amplitude (left), duration (middle), and latency (right) (n = 3 per group). h α-Bungarotoxin (BTX) staining of neuromuscular junctions (NMJs) in FDB muscles. The graph on the right shows the percentage of fragmented NMJs (n = 8 fields for Veh; n = 7 fields for Ino). i Relative hindlimb muscle weight (Sol, GAS, TA, and EDL muscles) normalized to body weight (n = 5 per group). j H&E staining (upper) and laminin immunostaining (lower, red) of TA muscles. The graph on the right shows the average CSA (µm²) (n = 7 fields for Veh; n = 6 fields for Ino). k Immunoblot analysis of the indicated phosphorylated (phospho-) or total proteins in GAS muscles from Veh- or Ino-treated aged mice. The graph below shows the ratio of phospho-to-total protein expression levels (n = 4 per group). l Immunostaining of IgM (green) and phalloidin (red) in TA muscles. The graph on the right shows the IntDen of the IgM signals (n = 16 fields per group). Data are presented as the mean ± SEM in panels (a, d, e, h, j and l); all other panels show the mean ± SD. Statistical significance was determined using two-way ANOVA (panel a and c), unpaired two-tailed Student’s t test (b, d, e, g, h, j, k and l) or one-way ANOVA (panel i). *p < 0.05, **p < 0.01, and ***p < 0.001

Compared with vehicle treatment, Ino treatment led to significant improvements in muscle strength and endurance exercise capacity (Fig. 4e). Given that neuromuscular dysfunction contributes to sarcopenia-related muscle weakness,^41,42 we assessed compound muscle action potentials (CMAPs) in the flexor digitorum brevis (FDB) muscles. Ino-treated aged mice displayed significantly increased CMAP amplitudes and shortened latencies, indicating improved neuromuscular transmission (Fig. 4f, g). Neuromuscular junction (NMJ) staining with alpha-bungarotoxin further revealed that compared with controls, FDB muscles treated with Ino markedly reduced the proportion of fragmented NMJs (Fig. 4h), suggesting that Ino protects against age-associated neuromuscular degeneration.

Organ weight analysis revealed no significant changes in liver, heart, kidney, eWAT, or BAT between the treatment groups (Supplementary Fig. 5b). However, hindlimb muscle weights were significantly greater in Ino-treated aged mice than in control mice (Fig. 4i), and the myofiber cross-sectional area in TA muscles was similarly increased (Fig. 4j). To explore the underlying mechanism, we examined anabolic signaling pathways critical for muscle maintenance. Ino treatment increased the phosphorylation of AKT1, MTOR, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and ribosomal protein S6 (RPS6), indicating that the activation of anabolic signaling likely contributes to muscle preservation in aged mice (Fig. 4k).

Furthermore, IgM immunostaining revealed reduced infiltration in Ino-treated muscles compared with Veh-treated controls, suggesting improved muscle integrity (Fig. 4l). In addition, gene expression analysis demonstrated significant upregulation of the expression of Myh1, a type IIx myofiber marker, in Ino-treated aged TA muscles, whereas the expression of markers for other types of myofiber remained unchanged (Supplementary Fig. 5c).

Collectively, these findings demonstrate that Ino treatment improved age-related muscular phenotypes, including reduced neuromuscular activity, loss of muscle mass, and deterioration of muscle function.

Ino-treated aged muscles exhibit altered expression of genes involved in metabolic pathways and immune responses

To elucidate the molecular mechanisms underlying the effects of Ino on aging muscle, we performed total RNA sequencing on GAS muscles from aged mice treated with vehicle or Ino. The PCA plot clearly shows separation between the vehicle- and Ino-treated samples along PC1, confirming consistent and reproducible transcriptional differences associated with treatment across biological replicates (Supplementary Fig. 5d). Transcriptomic analysis revealed candidate genes for pathway analysis of 987 genes (log2FC ≥ 0.75, p < 0.15), with 532 upregulated and 455 downregulated genes in Ino-treated muscles. A volcano plot highlights the top 20 most significantly affected genes (Fig. 5a). Gene Ontology (GO) enrichment mapping revealed two dominant functional clusters: immune response (78 nodes) and metabolic processes (54 nodes) (Fig. 5b). Ino treatment induced a directional shift, with metabolic process-related GO terms predominantly upregulated (red) and immune response-related terms largely downregulated (blue). This pattern was further validated by network visualization integrating the false discovery rate (FDR), term size and enrichment score (ES) via color, node size, and transparency, respectively, within a consistent network topology (Fig. 5c). The top GO terms with the highest normalized enrichment scores (NESs) in the metabolic process cluster included lipid localization, lipid metabolism, lipid homeostasis and fatty acid metabolic processes (Fig. 5d). Conversely, GO terms related to immune signaling, cytokine production, and positive regulation of the defense response were suppressed in aged Ino-treated muscles. A heatmap of core genes associated with at least three sub-GO terms revealed distinct clustering patterns of genes related to metabolic processes and immune response-related genes (Fig. 5e). Together, these results indicate that Ino treatment promotes metabolic reprogramming while suppressing inflammatory responses in aged muscles.

Fig. 5.

Ino-treated aged muscles exhibit altered expression of genes involved in metabolic pathways and immune responses. a RNA sequencing of GAS muscles treated with Ino compared with Veh is shown as a volcano plot. The x-axis shows the log2-fold change, and the y-axis shows the negative log10 P value. Approximately 984 genes passed the predefined threshold. Among these genes, 532 were upregulated, and 455 were downregulated. The twenty most strongly altered genes are labeled. The point color reflects the false discovery rate. b Gene Ontology enrichment results are presented as an enrichment map network. Each node represents a GO term and is colored according to the normalized enrichment score, with red indicating enrichment in Ino and blue indicating enrichment in Veh. Node size is proportional to the number of genes in the term, and edge width reflects the similarity coefficient based on gene overlap. c The same network is annotated with term-level statistics. Panels display the FDR q value, the gene set size, and the normalized enrichment score. d Representative sub-GO terms are summarized. The left circular layout shows metabolic processes enriched in Ino, and the right layout shows immune response terms enriched in Veh. The accompanying tables list the top terms ranked by normalized enrichment score. e Heatmaps showing the expression profiles of core genes within each cluster derived from the GO network, defined as genes overlapping across at least three subterms. qRT‒PCR analysis of the mRNA expression levels of genes related to metabolic processes (f) or immune responses (g) in GAS muscles from Veh- or Ino-treated aged male mice (n = 4 for Veh; n = 5 for Ino). The data are presented as the mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, and ***p < 0.001

To validate these transcriptomic findings, we performed qRT‒PCR analysis on RNA from vehicle- and Ino-treated aged muscles (Fig. 5f, g). The results confirmed the significant upregulation of genes related to metabolic processes and the marked downregulation of genes associated with immune responses in Ino-treated muscles compared with those in vehicle-treated muscles. These results indicate that the protective effects of Ino against age-related muscle wasting are mediated by enhanced metabolic pathway activity and attenuated inflammatory signaling.

Ino treatment improves the metabolic properties of aged muscles

To evaluate the effects of Ino on the metabolic properties of aged muscles, we first assessed mitochondrial oxidative and glycolytic activity using cytochrome c oxidase (COX)/SDH dual staining and glycerol-3-phosphate dehydrogenase (GPDH) activity assays in TA muscles. Compared with vehicle-treated controls, Ino-treated aged muscles exhibited markedly stronger COX-SDH staining and a trend toward increased GPDH activity, indicating enhanced oxidative and glycolytic metabolic capacity (Fig. 6a). Consistent with these findings, qRT‒PCR analysis revealed significant upregulation of mitochondrial genes in Ino-treated GAS muscles (Supplementary Fig. 6a). In parallel, the protein expression levels of OXPHOS complexes and myoglobin were significantly elevated in GAS muscles following Ino treatment (Fig. 6b; Supplementary Fig. 6b). Furthermore, the mtDNA content markedly increased in the GAS muscles, indicating enhanced mitochondrial biogenesis (Fig. 6c).

Fig. 6.

Ino treatment improves the metabolic properties of aged muscle and liver. a COX-SDH and GPDH enzyme activity staining of TA muscles. The graphs on the right show the quantification of COX- or GPDH-abundant areas (µm²; regions with strong enzyme activity; COX, brown color; GPDH, purple color) using Fiji (COX-SDH, n = 14 fields for Veh; n = 12 fields for Ino; GPDH, n = 10 fields for Veh; n = 9 fields for Ino). b Immunoblot analysis of total OXPHOS and myoglobin expression (n = 4 per group) in GAS muscles. c Relative ratio of mtDNA to nDNA (n = 4 per group) in GAS muscles. d JC-1 staining of live single myofibers isolated from EDL muscles. The graph on the right shows the ratio of the IntDen of JC-1 aggregates (red) to that of monomers (green) (n = 3 per group; 6 myofibers analyzed per individual). e MitoSOX (red) staining of live single myofibers isolated from EDL muscles. The graph on the right shows the IntDen of the MitoSOX signal (n = 3 per group; 6 myofibers analyzed per individual). f OCR was measured in mitochondria isolated from the GAS muscle of Veh- or Ino-treated aged mice (n = 3 per group; 10 μg of mitochondria per well). The indicated steps involved treatment with 1 mM ADP, 1.5 µM oligomycin, 2 µM FCCP, and 0.5 µM Rot/AA. g Quantification of mitochondrial respiration parameters derived from the data shown in panel f (n = 3 per group). h Immunoblot analysis of the indicated proteins in total lysates or Mito fractions of GAS muscles from Veh- or Ino-treated aged mice (n = 4 per group for total lysates; n = 3 per group for mito lysates). i BODIPY (green) and laminin (red) staining of TA muscles. The graph on the right shows the BODIPY-positive area (µm²) per field (n = 4 fields for Veh; n = 6 fields for Ino). Data are presented as the mean ± SEM in panels (a) and (i); all other panels show the mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test or two-way ANOVA (panel f). *p < 0.05, **p < 0.01, and ***p < 0.001

To directly evaluate mitochondrial functional capacity, we performed JC-1 staining on freshly isolated EDL myofibers from 30-month-old mice treated with vehicle or Ino for two weeks. Compared with vehicle-treated control myofibers, Ino-treated myofibers displayed a significantly greater JC-1 aggregate-to-monomer ratio, indicating enhanced mitochondrial membrane potential and function (Fig. 6d). Conversely, mitochondrial superoxide levels were markedly reduced in Ino-treated myofibers (Fig. 6e). In support of these findings, mitochondria were isolated from GAS muscles for respiration analysis (Fig. 6f, g). While basal respiration did not differ between groups, ATP-linked respiration, maximal respiration, and spare capacity generally increased in the Ino-treated group. These direct mitochondrial measurements indicate that Ino enhances not only the energy-producing capacity of mitochondria in aged muscle but also their metabolic flexibility.

Immunoblot analyses of total and mitochondrial lysates from vehicle- or Ino-treated aged GAS muscle demonstrated robust upregulation of key regulators of mitochondrial biogenesis and quality control, including PGC-1α and SIRT3, and increased expression of mitophagy-related proteins such as PTEN-induced kinase 1 (PINK1) and parkin RBR E3 ubiquitin protein ligase (PARK2) (Fig. 6h; Supplementary Fig. 6c, d). The expression of the autophagy markers microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (p62), as well as the lysosomal marker Lamp1, did not differ among the total lysates; however, analysis of the mitochondrial fractions revealed increased recruitment of LC3 to the mitochondria. These findings suggest that Ino is associated with mitochondrial remodeling, including increased mitochondrial content and the recruitment of mitophagy-related components.

Given the observed improvements in mitochondrial function, we next investigated whether Ino modulates lipid metabolism in aged tissues. BODIPY staining of TA muscles revealed substantially reduced intramuscular lipid droplet accumulation in Ino-treated aged mice compared with vehicle controls (Fig. 6i). Consistent with these findings, measurement of the lipid peroxidation marker malondialdehyde (MDA) in aged EDL muscle revealed a reduction in MDA levels in the Ino-treated group (Supplementary Fig. 6e). Notably, a similar decrease in MDA levels was observed following Ino treatment in the DEX-induced muscle atrophy model (Supplementary Fig. 6f). Similarly, histological analyses of liver sections revealed a marked reduction in hepatic fat deposition in Ino-treated mice, which was corroborated by BODIPY staining of hepatic tissues (Supplementary Fig. 6g).

At the transcriptional level, qRT‒PCR analyses demonstrated significant downregulation of lipogenic genes, including sterol regulatory element binding transcription factor 1 (Srebf1) and apolipoprotein E (Apoe), in Ino-treated aged livers compared with those in vehicle controls (Supplementary Fig. 6h). Ino treatment also mildly but significantly increased LXRβ (Nr1h2) expression, whereas LXRα (Nr1h3) levels remained unchanged. Consistently, immunoblot analyses revealed a substantial decrease in the hepatic lipid accumulation regulators SREBP1c and stearoyl-CoA desaturase 1 (SCD-1), accompanied by increased phosphorylation of acetyl-CoA carboxylase (ACC), indicating suppressed de novo lipogenesis (Supplementary Fig. 6i).

Collectively, these findings demonstrate that Ino treatment enhances mitochondrial function and promotes metabolic remodeling by simultaneously increasing mitochondrial biogenesis, mitophagy, and lipid metabolism in aged muscle and liver.

Ino enhances SIRT3/PGC-1α activity and mitochondrial function through LXRβ activation

Because Ino enhances mitochondrial function and metabolic pathways, we next explored its molecular targets. SwissTargetPrediction identified 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) and LXRs as top candidates, but Ino had no effect on HMG-CoA reductase activity. We therefore focused on LXRs, particularly LXRβ, which is consistent with a recent report that Ino activates LXRβ and reduces neuroinflammation in an Alzheimer’s disease model.⁴³

To directly assess whether LXRβ is required for the effects of Ino on muscle metabolism and mitochondrial function, we transfected C2C12 myoblasts with siRNA targeting LXRβ. Among the tested siRNAs, siLXRβ-#3 reduced LXRβ expression by ~50% (Supplementary Fig. 7a). Knockdown of LXRβ expression significantly reduced the expression of target genes related to lipid metabolism and glucose metabolism, whereas the expression of mitochondrial genes such as Pparg and Ppargc1a remained unaffected under basal conditions (Supplementary Fig. 7b).

When LXRβ-depleted cells were treated with Ino, the Ino-induced upregulation of Srebf1 and Apoe expression was significantly reduced. Importantly, the ability of Ino to increase the expression of Pparg, Ppargc1a and Sirt3 was markedly attenuated in LXRβ-depleted cells compared with that in vehicle-treated cells (Fig. 7a). These findings strongly suggest that LXRβ plays a crucial role in mediating the effects of Ino, particularly in regulating mitochondrial function via the SIRT3/PGC-1α pathway.

Fig. 7.

Ino enhances SIRT3/PGC-1α activity and mitochondrial function through LXRβ activation. a qRT‒PCR analysis of the mRNA expression of the indicated genes in C2C12 cells transfected with siRNA targeting LXRβ (siLXRβ) or scrambled RNA (negative control). Cells were treated with Veh or 1 μM Ino for 24 h. Rn18s was used as an internal control (n = 3 per group). b BODIPY (green) staining of C2C12 cells treated with PA in combination with Veh or Ino. The graph on the right shows the IntDen of the BODIPY signal (n = 3 fields per group, normalized by cell number). c qRT‒PCR analysis of genes related to mitochondrial metabolism in C2C12 cells under the indicated conditions (n = 3 per group). d JC-1 staining of C2C12 cells under the indicated conditions. The graph on the right shows the relative IntDen ratio of JC-1 aggregates (red) to monomers (green) (n = 15 fields per group). e TMRM staining of C2C12 cells under the indicated conditions. The graph below shows the relative TMRM fluorescence intensity (Ex/Em = 548/573 nm) (n = 11 per group). Unstained cells were used as a negative control. f MitoTracker (green) and MitoSOX (red) staining of C2C12 cells under the indicated conditions. The graphs on the right show the relative IntDen of MitoSOX normalized to the MitoTracker signal (upper), mean mitochondrial branch length (middle), and total mitochondrial branch length (lower) (n = 11 wells per group). g qRT‒PCR analysis of the expression of antioxidant-related genes (Gpx1 and Idh1) in C2C12 cells under the indicated conditions (n = 3 per group). The indicated steps involved treatment with 1.5 µM oligomycin, 3 µM FCCP, and 0.5 µM Rot/AA. h OCR values in C2C12 cells under the indicated conditions were normalized to the mtDNA copy number (mtDNA/nDNA) and multiplied by 10³ for visualization (n = 3 per group). Data are presented as the mean ± SEM in panel (e); all other panels show the mean ± SD. Statistical significance was determined using one-way ANOVA (panels b–g) or two-way ANOVA (panels a and h). *p < 0.05, **p < 0.01, and ***p < 0.001

We next examined the protective effects of Ino against lipotoxicity. C2C12 myoblasts were treated with PA along with vehicle or Ino. Compared with vehicle treatment, PA exposure suppressed the expression of genes related to lipid homeostasis (Supplementary Fig. 7c). Ino cotreatment attenuated this suppression, except for that of fatty acid synthase (Fasn). In addition, Nr1h2 expression was significantly elevated by Ino cotreatment. Consistent with this disrupted lipid metabolism, PA exposure led to significant lipid droplet accumulation, as visualized by BODIPY staining, and this accumulation was greatly reduced by Ino cotreatment (Fig. 7b).

PA treatment reduced the expression of mitochondrial genes, and Ino cotreatment alleviated this suppression (Fig. 7c). Consistent with the increase in the expression of genes related to mitochondria, Ino treatment increased mitochondrial activity, as measured by JC-1 and TMRM staining (Fig. 7d, e). Furthermore, compared with vehicle treatment, Ino treatment partially preserved the PA-induced reduction in mitochondrial branch length, attenuated mitochondrial fragmentation and oxidative stress, and restored the expression of glutathione peroxidase 1 (Gpx1) and isocitrate dehydrogenase 1 (Idh1) (Fig. 7f, g). In line with these findings, cells were treated with PA under Veh or Ino conditions, and mitochondrial respiration was assessed (Fig. 7h; Supplementary Fig. 7d). While basal and ATP-linked respiration did not differ between the groups, maximal respiration and spare capacity were increased in the Ino-treated cells. These results suggest that Ino maintains basal energy production while enhancing mitochondrial responsiveness to PA-induced metabolic stress.

Taken together, these data demonstrate that Ino enhances the activity of the SIRT3/PGC-1α pathway via LXRβ, which contributes to improved lipid metabolism, enhanced mitochondrial function, and antioxidative responses.

Ino induces SIRT3 expression via LXRβ together with PPARδ

To further elucidate the mechanism of Ino activity, we compared its regulatory effects with those of the well-known synthetic LXR agonist T0901317 (T090). Consistent with previous findings, both Ino and T090 significantly induced the expression of lipid metabolism-related genes in muscle cells (Fig. 8a). However, unlike T090, Ino robustly upregulated key mitochondrial regulatory genes, highlighting its distinct role in mitochondrial biogenesis and functional enhancement (Fig. 8b). In line with these findings, these transcriptional changes, Ino markedly increased the mtDNA content, whereas T090 had no comparable effect (Fig. 8c). Together, these findings suggest that Ino activates LXRβ beyond its canonical lipid regulatory role, extending its influence to mitochondrial homeostasis.

Fig. 8.

Ino induces SIRT3 expression via LXRβ together with PPARδ. qRT‒PCR analysis of genes related to lipid metabolism (a) and mitochondria (b) in C2C12 cells treated with Veh, 1 µM Ino, or 1 µM T090 for 24 h. Rn18s was used as a loading control (n = 3 per group). c Relative ratio of mtDNA to nDNA in C2C12 cells under the indicated conditions (n = 3 per group). d Immunoblot analysis of total OXPHOS proteins in C2C12 cells under the indicated conditions. β-tubulin served as a loading control (n = 3 per group). e Immunostaining of LXRβ (red) in C2C12 cells treated with Veh or Ino. The colocalization of LXRβ with the nucleus is shown in white. Nuclei were counterstained with DAPI (blue). The graph on the right shows the relative IntDen of the LXRβ signal (n = 9 fields for Veh; n = 8 fields for Ino). f Immunoblot analysis of LXRβ protein levels in the nuclear (Nuc) and cytoplasmic fractions of C2C12 cells treated with Veh or 1 µM Ino. β-Tubulin (a cytosolic marker) or Lamin B1 (LMNB1, a nuclear marker) served as loading controls (n = 2 per group). g Schematic representation of putative LXRβ-binding elements (region 1 and region 2) within the 1 kb promoter region of the mouse Sirt3 gene. The schematic was created using BioRender.com. h ChIP assay for LXRβ occupancy at the putative binding regions of the Sirt3 promoter in C2C12 cells treated with Veh or 1 µM Ino. IgG was used as a negative control, and its background value was subtracted (n = 3 per group). i ChIP assay for LXRβ binding at the Sirt3 promoter in C2C12 cells treated with Veh, 1 µM Ino, or 1 µM T090 for 24 h (n = 3 per group). j Immunoblot analysis and quantification of LXRβ and PPARδ protein expression in GAS muscles from 20-month-old mice treated with Veh or Ino (n = 4 per group). k Immunoprecipitation analysis with a rabbit IgG or LXRβ antibody followed by immunoblotting for LXRβ and PPARδ in C2C12 cells. l Immunoprecipitation analysis with anti-LXRβ antibody followed by immunoblotting for LXRβ and PPARδ in C2C12 cells treated with Veh or 1 µM Ino. m Immunoprecipitation analysis with anti-LXRβ antibody followed by immunoblotting for LXRβ and PPARδ in GAS muscles from 20-month-old mice treated with Veh or Ino. The left graph represents the relative Co-IP ratio obtained by quantifying the Co-IP signals of PPARδ and LXRβ, where PPARδ was normalized to LXRβ (n = 3 per group). n ChIP assay for PPARδ binding to the Sirt3 promoter in C2C12 cells (n = 3 per group). o ChIP assay for PPARδ binding at the Sirt3 promoter in C2C12 cells transfected with scrambled RNA or siLXRβ and treated with either vehicle (Veh) or 1 μM Ino for 24 h (n = 3 per group). Data are presented as the mean ± SEM in panel (h); all other panels show the mean ± SD. Statistical significance was determined using one-way ANOVA (a–c, i, n and o) or an unpaired two-tailed Student’s t test (e, h, j and m). *p < 0.05, **p < 0.01, and ***p < 0.001

In support of this notion, Ino treatment significantly increased total OXPHOS protein levels, indicating enhanced mitochondrial content and oxidative capacity, whereas T090 failed to elicit a similar effect (Fig. 8d). Collectively, these findings indicate that unlike previously reported LXR agonists, which mainly regulate lipid metabolism, Ino exerts dual regulatory effects by alleviating lipotoxicity while simultaneously preserving mitochondrial integrity.

To investigate the molecular basis of this regulation, we evaluated LXRβ activation and subcellular localization. Immunofluorescence and nuclear fraction analyses revealed that Ino treatment of C2C12 cells for 24 h increased overall LXRβ fluorescence intensity and promoted its nuclear accumulation (Fig. 8e, f). Consistent with these findings, LXRβ protein levels were elevated in both nuclear and cytosolic compartments, suggesting enhanced nuclear activity.

Next, we examined whether Ino-induced Sirt3 expression is mediated by direct binding of LXRβ to the Sirt3 promoter. Sequence analysis revealed two putative LXRβ-binding motifs within the upstream regulatory region of the Sirt3 promoter (-816/-806; -504/-494) (Fig. 8g). Chromatin immunoprecipitation (ChIP) assays confirmed LXRβ occupancy at both sites, while region 2 showed stronger binding affinity (Fig. 8h). Notably, Ino treatment strongly increased LXRβ binding at region 2, whereas T090 failed to induce significant promoter occupancy (Fig. 8i), indicating that Ino regulates Sirt3 expression via a unique LXRβ-dependent mechanism.

Considering the established role of PPARδ in promoting fatty acid oxidation and mitochondrial biogenesis in skeletal muscle,⁴⁴ we hypothesized that LXRβ and PPARδ potentially interact to mediate Ino-induced mitochondrial reprogramming. In aged GAS muscle, Ino treatment increased PPARδ expression in parallel with LXRβ expression, suggesting a possible interaction (Fig. 8j). Notably, PPARδ was coprecipitated with anti-LXRβ antibodies in C2C12 cell lysates, and Ino treatment further enhanced this interaction (Fig. 8k, l). Consistently, in aged muscle, Ino treatment increased PPARδ expression in parallel with LXRβ expression, suggesting a possible interaction (Fig. 8m). Furthermore, ChIP assays using a PPARδ-specific antibody in C2C12 cells treated with vehicle, Ino or T090 for 24 h demonstrated that PPARδ is recruited to region 2 of the Sirt3 promoter, with Ino inducing significantly stronger recruitment than T090 (Fig. 8n). Importantly, PPARδ binding was markedly diminished in LXRβ-depleted cells (Fig. 8o), demonstrating that Ino-mediated SIRT3 upregulation requires coordinated activation of the LXRβ-PPARδ axis. However, Ino treatment did not substantially alter the nuclear localization of PPARδ, suggesting that its effect is primarily dependent on LXRβ (Supplementary Fig. 8a). Comparisons of young and aged GAS muscles at baseline revealed that LXRβ expression was reduced in aged muscle, whereas PPARδ expression and the LXRβ–PPARδ interaction remained unchanged (Supplementary Fig. 8b, c). These results indicate that aging alone does not alter this interaction and suggest that Ino potentially modulates the binding efficiency of LXRβ to PPARδ, thereby regulating SIRT3 expression and mitochondrial function.

In summary, long-term Ino administration mitigates aging- or DEX-induced muscle wasting by activating the LXRβ/SIRT3/PGC-1α pathway and enhancing lipid metabolism and mitochondrial function, thereby improving muscle performance and metabolic health.

Discussion

Sarcopenia is an age-associated disorder characterized by progressive decreases in skeletal muscle mass, strength, and performance.⁶ Despite increasing evidence indicating that mitochondrial dysfunction and disrupted lipid metabolism are involved in muscle aging, the mechanisms connecting these processes remain incompletely understood. In the present study, we demonstrate that Ino effectively preserves muscle mass and function by improving mitochondrial efficiency and promoting energy metabolism adaptability. Ino enhanced oxidative phosphorylation capacity and increased the expression of genes involved in mitochondrial biogenesis, lipid metabolism, and glucose utilization. These beneficial effects occurred without altering muscle fiber-type composition, suggesting that Ino primarily acts through metabolic remodeling while preserving muscle structural integrity. Furthermore, we show that Ino restores LXRβ, SIRT3, and PGC-1α expression, improves mitochondrial function, and reduces oxidative stress in aged muscle. Collectively, our findings identify a previously unrecognized role for LXRβ in skeletal muscle metabolic regulation and suggest that the LXRβ/SIRT3/PGC-1α pathway contributes to the protective effects of Ino.

Our findings are consistent with those of previous studies demonstrating that SIRT3 and PGC-1α are essential regulators of mitochondrial biogenesis, oxidative metabolism, and antioxidant defense in skeletal muscle.^21,22,45 Reduced expression of these regulators has been reported in aging muscle and is associated with impaired oxidative capacity, defective stress responses, and anabolic resistance.^24,27 In agreement with these observations, the aged muscles in our study exhibited mitochondrial dysfunction, oxidative stress, and lipid accumulation, all of which improved following Ino treatment. Previous studies have also shown that exercise and nutritional interventions enhance mitochondrial function through the activation of PGC-1α-SIRT3-dependent pathways.^44–46 Therefore, the metabolic adaptations induced by Ino resemble the physiological responses associated with improved mitochondrial fitness. Notably, the coordinated restoration of SIRT3 and PGC-1α expression observed in our study suggest that Ino not only improves mitochondrial function but also promotes broader metabolic remodeling in aging skeletal muscle. Enhanced mitochondrial biogenesis and oxidative metabolism are likely to increase ATP production, improve fatty acid oxidation, and reduce excessive reactive oxygen species generation, thereby preserving cellular integrity and contractile function. Furthermore, these improvements may enhance metabolic flexibility, enabling aged muscle to respond more effectively to energetic stress while limiting lipid-induced metabolic dysfunction. In addition, our findings extend previous reports describing interactions between LXR and PPAR signaling pathways and provide evidence that these pathways may cooperate to regulate skeletal muscle metabolism. Given that PPAR activation has been closely linked to mitochondrial oxidative capacity and lipid utilization, the simultaneous activation of LXRβ and PPAR signaling may represent an integrated regulatory mechanism underlying the beneficial metabolic effects of Ino. Together, these results support the concept that the restoration of mitochondrial and metabolic homeostasis is an effective strategy for attenuating age-associated muscle decline.

Several observations from the present study provide additional mechanistic insight. Unlike the synthetic LXR agonist T090, which predominantly induces the expression of lipid-related genes without substantially improving mitochondrial function, Ino increased the mtDNA content, OXPHOS protein expression, and mitochondrial abundance. ChIP assays demonstrated that Ino promoted LXRβ binding to the Sirt3 promoter and selectively enhanced the recruitment of PPARδ to this region, effects that were abolished following LXRβ depletion. These findings suggest that Ino induces a distinct transcriptional program involving coordinated LXRβ-PPARδ signaling rather than simply functioning as a conventional LXR agonist. Given the established role of PPARδ in promoting fatty acid oxidation, mitochondrial biogenesis, and oxidative remodeling, the activation of this pathway may contribute to the metabolic benefits observed following Ino treatment.^44,45,47 The selective recruitment of PPARδ to the Sirt3 promoter further suggests that Ino facilitates transcriptional crosstalk between lipid-sensing nuclear receptors and mitochondrial regulatory networks. This coordinated mechanism may enable a more balanced metabolic response by simultaneously enhancing lipid utilization, preserving mitochondrial integrity, and limiting oxidative damage, thereby supporting sustained muscle function during aging. Moreover, Ino improved hepatic metabolic profiles and attenuated lipid accumulation, suggesting that modulation of the LXRβ-PPARδ pathway may confer systemic metabolic benefits beyond skeletal muscle.⁴⁸ These systemic effects raise the possibility that Ino improves inter-organ metabolic communication, which may indirectly contribute to the preservation of skeletal muscle homeostasis and overall metabolic health in aged animals. Nevertheless, several limitations should be acknowledged. Although our data identify LXRβ as an important mediator of Ino activity, additional studies using tissue-specific genetic models will be needed to define the precise contribution of individual downstream pathways and to determine whether additional molecular targets participate in the observed effects. Future investigations should also evaluate the long-term efficacy, and translational potential of Ino in preclinical models and ultimately in human studies.

The favorable metabolic profile of Ino highlights its translational potential. Recent studies have demonstrated that Ino selectively binds LXRβ with minimal activity toward LXRα.⁴³ Consistent with this selectivity, prolonged administration of Ino did not induce abnormalities in plasma lipid profiles, in contrast to conventional LXR agonists such as T090 and GW3965, which activate both LXRα and LXRβ and frequently induce hepatic lipogenesis and hypertriglyceridemia, thus limiting their clinical applicability.^49,50 Moreover, compared with endogenous oxysterols (EC50 = 2–10 μM) and several previously reported synthetic agonists, Ino has a remarkably low half maximal effective concentration (EC50, <10 nM) for LXRβ activation.⁴³ This high potency, together with its receptor selectivity, may allow effective modulation of metabolic pathways at substantially lower doses, thereby reducing the likelihood of adverse effects associated with non-selective LXR activation. Such pharmacological characteristics make Ino an attractive candidate for the long-term management of chronic age-related disorders that require sustained therapeutic intervention. In addition to skeletal muscle, Ino also improved hepatic lipid metabolism and prevented lipid accumulation, suggesting broader systemic metabolic effects. These findings further indicate that Ino may improve whole-body metabolic homeostasis by coordinating lipid utilization and mitochondrial function across multiple metabolically active tissues. Such systemic metabolic improvements could provide additional benefits of insulin sensitivity, energy balance, and age-associated metabolic dysfunction. Because mitochondrial dysfunction and lipid dysregulation are common features of aging and metabolic diseases, modulation of the LXRβ/SIRT3/PGC-1α pathway may represent a promising therapeutic strategy that extends beyond sarcopenia. Future clinical studies will be necessary to determine whether these preclinical benefits can be translated into safe and effective therapeutic interventions for aging populations.

In summary, our findings identify Ino as a potent and selective activator of LXRβ signaling that preserves muscle mass and function through activation of the LXRβ/SIRT3/PGC-1α pathway and coordinated LXRβ-PPARδ signaling. Unlike conventional LXR agonists, Ino improves mitochondrial efficiency, restores lipid homeostasis, and mitigates oxidative stress without inducing adverse lipid metabolic effects. These findings establish a previously unrecognized connection between lipid sensing and mitochondrial regulation in skeletal muscle and support further investigation of Ino as a therapeutic candidate for sarcopenia and other age-related metabolic disorders.

Materials and methods

Animal experiments

Animal studies were conducted using three different C57BL/6 male mouse models: young (3 months old), aged (≥20 months old), and dexamethasone (DEX, Sigma-Aldrich, D2915)-induced muscle atrophy. Ino was orally administered at a dose of 0.35 mg/kg, prepared in a vehicle composed of 40% PEG (Sigma, 25322-68-3) and 5% Tween 80 (Glentham Life Sciences, 9005-65-6) in 0.9% normal saline. The DEX-induced atrophy model was established by intraperitoneal injection of DEX (20 mg/kg daily) in 9-week-old mice for 10 days, together with oral administration of vehicle or Ino for 10 days.

Muscle function was evaluated by grip strength measurement, treadmill running performance, and electromyography (EMG), with all experiments conducted in a blinded manner. Grip strength was measured on the forelimbs as previously described.⁵¹ Each mouse underwent a total of four consecutive grip strength measurements using grip strength meter (Bioseb, 76-0483), and the average of these values was defined as the individual mouse’s grip strength. Treadmill testing was age-dependent: young mice were tested on an inclined treadmill (10% slope; Columbus Instruments Exer-6M Treadmill) to increase muscular load, whereas aged mice were tested on a flat treadmill. Running started at 8 m/min and increased by 1 m/min every 2 min. Compound Muscle Action Potentials (CMAPs) and EMG responses were recorded using the RS-EMG-SA-STIM Small Animal EMG System with Stimulation (iWorx Systems, Inc.). Measurements were obtained by stimulating the sciatic nerve and recording compound muscle action potentials from FDB muscle prior to tissue harvest at sacrifice. Serum was collected at the time of sacrifice for the determination of TG (Elabscience, E-BC-K261-S) and FFA (Invitrogen, EEA017).

Blood glucose levels were measured following a 16 h overnight fast, during which mice had free access to water. Small incisions were made on the tail vein using a razor blade, and blood glucose was measured at least twice per mouse using glucometer (i-sense, GM01JAC) and test strips.

All procedures were approved by the Institutional Animal Care and Research Advisory Committee at Sungkyunkwan University School of Medicine Laboratory Animal Research Center (SKKUIACUC2025-03-39-1) and complied with institutional ethical regulation. In addition, third-party validation for Ino efficacy in young and DEX models was conducted under an approved protocol at Gachon University (LCDI-2021-0084).

Statistical analysis

To analyze statistical differences, an unpaired Student’s t test was used for comparisons between two groups. For comparisons among three or more groups, one-way or two-way ANOVA was applied as appropriate. Two-way repeated-measures ANOVA was performed when longitudinal measurements were obtained from the same subjects, as shown in Fig. 4a. The statistical tests used are indicated in each figure legend. Values are expressed as mean ± standard deviation (SD) or standard error of the mean (SEM).

The detailed materials and methods are described in the Supplementary Materials.

Author contributions

C.L.Y., J.H.B., Y.E.L., S.J.L., G.U.B., J.S.K. conceived and designed the project. C.L.Y., J.H.B., J.K., J.K., J.P., T.A.V., J.J., S.P., M.J. performed the experiments. Y.J. carried out bioinformatics analysis. C.L.Y., J.H.B., Y.J., M.J.P., I.Y.K., Y.S.L., G.U.B. and J.S.K. analyzed data. C.L.Y., J.H.B., G.U.B. and J.S.K. wrote the manuscript. All authors have read and approved the article.

Data availability

The raw bulk RNA-seq data generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE335984. The lists of differentially expressed genes, including their fold-change values and associated analysis results, are provided in Supplementary Table S3. All other data supporting the findings of this study are available from the corresponding author (J.S.K.) upon reasonable request.

Competing interests

C.L.Y., J.H.B., and S.J.L. have filed a patent entitled “COMPOSITION COMPRISING INOTODIOL FOR PREVENTION OR TREATMENT OF MUSCULAR DISEASE” (PCT/KR2021/015026). G.U.B. and J.S.K. are founders of AniMusCure Inc. and hold an ownership equity interest in the company. These arrangements have been reviewed and approved by Sookmyung University (G.U.B.) and Sungkyunkwan University (J.S.K.) in accordance with their conflict of interest policies.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41392-026-02935-7.